The long-term objective of this project is to understand how insulin regulates the effects of amphetamine (AMPH) that are related to its abuse. Drug abuse remains a significant public health issue with important economic and social ramifications. With regard to AMPH, development of pharmacotherapies is limited by a severe lack of information on how AMPH works at the molecular level. Dopamine (DA) transporters (DATs) largely control DA clearance from the synapse and are one important site of action for psychostimulants such as cocaine and AMPH. AMPH attenuates DA clearance efficiency by acting on DAT, thereby increasing synaptic DA levels and enhancing DA transmission. This is believed to be a primary effect contributing to AMPH abuse. Very little is known about the molecular mechanisms by which AMPH regulates DAT activity and how that regulation translates to reinforcing effects. This project will combine biochemistry, confocal microscopy and behavior to elucidate the mechanisms involved in the insulin regulation of: a) AMPH-induced DAT cell surface redistribution, and b) self administration of AMPH. The key issues that will be addressed include how insulin regulates DAT trafficking and whether AMPH causes DAT cell surface redistribution, by interfering with the insulin signaling cascade. The trafficking process affects DAT-mediated DA reuptake, ultimately regulating extracellular concentrations of DA that contribute to behavioral actions. Therefore, behavioral studies of AMPH self administration will be conducted in rats made diabetic by pharmacologic means in order to test the hypothesis that the reinforcing effects of AMPH are regulated by insulin. Our experimental plan links trafficking of the DAT to the abuse-related behavioral effects of AMPH. The proposed studies address the following Specific Aims: 1) characterize the acute insulin regulation of DA transport; 2) determine whether chronic depletion of insulin regulates DA uptake in rat striatum; and 3) assess whether chronic depletion of insulin selectively modifies the reinforcing effects of AMPH. This proposal seeks to identify molecular mechanisms for AMPH that should provide new cellular targets for substance abuse therapies.